Browsing by Subject "Flow Cytometry"
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Item Molecular Characterization of the Human Alloimmune Response(2009-11-18) Smith, Melody; Collins, RobertWhen T lymphocytes from two individuals are placed in a mixed lymphocytic reaction (MLR) and allowed to stimulate each other a powerful proliferative response is elicited, reflecting the degree of incompatibility at the major histocompatibiltiy loci. Such in vitro proliferations translate in vivo into graft rejection of stem cell and solid organ transplants and to graft versus host disease (GvHD) in recipients of allogeneic stem cell transplants. Defining the clonal T cell responses elicited in an MLR between two individuals is complicated by the multiplicity of T cell HLA- antigen interactions. We used flow cytometry to investigate the MLR between mismatched individuals to better characterize responder T cell proliferation and the subsets involved in the alloresponse. After observing proliferation in the setting of a completely mismatched one-way MLR and ensuring that the design for the MLR was viable, we sought to characterize clonal T cell responses in a simple system. We attempted to elicit T cell responses to autologous antigen-presenting cells (APC) transfected with a single mismatched HLA-A2 molecule. We failed to detect any alloresponding T cells using a flow cytometric system, except when dendritic cells were used. We then used an HLA-A2 negative responder pre-stimulated in an MLR with an HLA-A2 positive individual for re-challenge by the responder?s own APC transfected with an HLA-A2 plasmid. In this way, we sought to identify an expanding HLA-A2 specific T cell population which could be further characterized by T cell receptor cloning. Nevertheless, we failed to detect an alloimmune response in this T cell line stimulated with HLA-A2 transfected APCs three times over a 21- day culture period. These results suggest that either the frequency of alloreactive T cells was below the limit of detection by these methods or that the particular stimulator-responder pairs used in these experiments were non-reactive. Future work will extend the study to more HLA mismatched stimulator-responder pairings to better define the characteristics of alloreactive T cells.Item Transcriptional Profiling of Early Cardiac Development(2005-08-11) Masino, Amanda M.; Garry, Daniel J.Cardiogenesis is an intricate process that spans nearly the entire developmental history of the mammal. A thorough comprehension of the molecular interactions that direct cardiac development will improve our ability to understand and treat cardiovascular diseases. The earliest stages of heart development are of particular interest because it is during these stages that patterning of cardiac structure appears critical. Traditionally, studies of these early developmental processes have been conducted on chicken, fish, or frog embryos due to ease of manipulation. More recently, however, the wide availability of tools designed to manipulate and isolate cell populations in the murine embryo, together with the advent of technologies for studying gene expression at the transcriptome level, have allowed for detailed study of these events in mammalian systems. I applied transgenic labeling and flow cytometry to isolate cardiac cell populations from early mouse embryos. Transcriptome analysis of these cell populations allowed me to characterize unique gene expression patterns and identify novel genetic pathways relevant to cardiac development. I undertook three interrelated studies. First, I completed a survey of the transcriptome of the developing heart at the cardiac crescent, linear heart tube, and looped heart tube stages to describe molecular signatures in the developing heart. I defined gene expression patterns unique to the cardiac crescent and looped heart, and identified novel markers of the developing heart. The second study focused on defining the transcriptional network of a key cardiac regulator, Nkx2.5. I identified transcripts misexpressed during the early development of Nkx2.5 null embryos, including the Nkx2.5 transcriptional targets ETSrelated transcription factor 71 and vascular cell adhesion molecule. Lastly, I characterized Nkx2.5-dependent left-right patterning in the cardiac crescent that revealed early specification of chamber myocardial lineages. In conclusion, these studies have elucidated the molecular profile of the developing heart and provided novel insights into the earliest stages of cardiac development.